1 Field of the Invention
The present invention relates to methods for controlling endosperm size and development, and seed viability in plants. The invention also relates to nucleic acid constructs for use in such methods, as well as modified plants per se.
2. Related Art
Yield in crop plants where seed is the harvested product is usually defined as weight of seed harvested per unit area (Duvick, 1992). Consequently, individual seed weight is regarded as a major determinant of yield. Most monocotyledonous plants e.g. maize, wheat, (see Esau, 1965) produce albuminous seeds—that is, at maturity they contain a small embryo and a relatively massive endosperm. Consequently, in monocotyledonous plants, the endosperm represents a significant component of seed yield. Endosperms accumulate and store diverse substances, including starch, proteins, oils and fats.
Therefore, in monocotyledons increasing the size of the endosperm or its ability to accumulate storage products is likely to increase individual seed weight and perhaps total yield.
Endosperms are utilised commercially in diverse ways, either indirectly as part of the whole seed or directly following their purification, or the purification of certain of their constituents. Hence endosperms may represent either a proportion or the entire commercial value of a crop. Examples of indirect usage include fodder maize and whole wheat flour. An example of direct usage of the complete endosperm is in the production of white flour for bread-making. Finally, maize oil represents an example of the utilisation of a constituent of the endosperm, but there are many others.
In contrast to monocotyledons, most dicotyledonous plants, e.g. oil seed rape, soybean, peanut, Phaseolus vulgaris (e.g. kidney beans, white beans, black beans), Vicia faba (broad bean), Pisum sativum (green pea), Cicer aeietinum (chick pea), and Lens culinaris (lentil) produce exalbuminous seeds—that is, mature seeds lack an endosperm. In such seeds the embryo is large and generally fills most of the volume of the seed, and accounts for almost the entire weight of the seed. In exalbuminous seeds the endosperm is ephemeral in nature and reaches maturity when the embryo is small and highly immature (usually heart/torpedo stage). Commonly embryo development depends on the presence of the endosperm, which is generally accepted to act as a source of nutrition for the embryo.
Scott et al (1998) showed that the size of the endosperm in terms of the number of endosperm cells at maturity in the dicotyledonous plant Arabidopsis thaliana, a close relative of oil seed rape (Brassica napus), is positively correlated with the weight of the mature seed. Plants that developed seeds with 80% smaller endosperms (average=80 nuclei) compared to controls (mean of 2x-2x (diploid plant crosses) and 4x-4x (tetraploid plant crosses)=400 nuclei) produced seeds that were 46% smaller (in weight terms=14 μg) than the controls (mean of 2x-2x and 4x-4x=30 μg). In contrast, plants that developed seeds with 160% bigger endosperms (average 640 nuclei) compared to controls (mean of 2x-2x and 4x-4=400 nuclei) produced seeds that were 180% larger (in weight terms=54 μg) than the controls (mean of 2x-2x and 4x-4x=30 μg). Arabidopsis seed in common with most other dicotyledonous seed is composed almost entirely of embryo. Hence the change in seed weight is almost completely due to a change in embryo weight.
Consequently, modifying endosperm size, in terms of the number of cells at maturity, has a dramatic impact on seed weight in seeds that do not contain endosperm at maturity. Without being bound by the following, one reasonable hypothesis is that a larger endosperm accumulates a greater quantity of reserves from the seed parent, perhaps by acting as a stronger “sink”. These reserves then provide more resources for utilisation by the growing embryo, resulting in a larger seed. Alternative mechanisms might operate, however.
The seeds of dicotyledons, like those of monocotyledons are utilised in diverse ways. For example, pulses such as soybean, peanut, Phaseolus vulgaris (e.g. kidney beans, white beans, black beans), Vicia faba (broad bean), Pisum sativum (green pea), Cicer aeietinum (chick pea), Lens culinaris (lentil) are important world crops that are used directly for animal and human consumption. Seeds of oil rape, sunflower and linseed are processed to produce oils.
Clearly, despite the differences in the structure of monocot and dicot seeds, particularly with respect to the presence or absence of endosperm in mature seeds, the size of the endosperm is an important factor in determining individual seed weight, and therefore potentially total crop yield in plants where seed is the economic harvest. Indeed, Hannah and Greene (1998) showed that maize seed weight is dependent on the amount of endosperm ADP-glucose pyrophosphorylase, the enzyme responsible for supplying substrate for starch synthesis.
However, there is some evidence that an increase in seed weight is associated with a reduction in seed number in many breeding populations. Consequently, increasing individual seed size may not result in an increase in total yield. While maize breeding programmes have been successful and genetic improvement has played a significant role in increased maize yields, the genetic component to yield has led to only a doubling of this parameter since the 1930s (Duvick, 1992). The increase in maize yields is currently less than 1% per year.
The genetic basis for the resistance to increased seed weight encountered in conventional breeding programmes is not understood. However, Giroux et al (1996) showed that a single gene mutation in the endosperm specific gene shrunken2 of maize resulted in a seed weight increase of 11-18% without a reduction in seed number. This suggests that yield improvements are possible in a plant with a long history of intensive and successful breeding efforts, and may therefore be generally achievable. Similarly, Roekel et al. (1998) showed that introduction of the tzs gene into Brassica napus results in a significant increase in seed yield accounted for by increased seed number per silique and increased seed weight.
There is also evidence that seed size (weight) is positively correlated with a number of components of “seed quality” such as percent germination (Schaal, 1980: Alexander and Wulff, 1985; Guberac et al, 1998); time to emergence (Winn, 1985; Wulff, 1986); durability (survival under adverse growing conditions) (Krannitz et al, 1991; Manga and Yadav,1995); growth rate (Marshall, 1986) and yield (Guberac et al, 1998). Seed quality is an important factor in the cost of production of commercial seed lots since these must be tested before sale. Consequently, increasing total seed weight, even without increases in total seed yield may have economic benefits through improvements in seed quality.
We have recently demonstrated (Scott et al., 1998) that hybridising Arabidopsis plants of different ploidies has reproducible and dramatic effects on the weight of progeny seed. For example, an interploidy cross between a diploid (2x) seed parent and a tetraploid (4x) pollen parent (2x-4x) results in seed which is 240% larger than 2x-2x seed. Conversely, 4x-2x crosses result in a reduced seed size (60% of 2x-2x). Analysis of endosperm development in these F1 seed reveals a clear correlation between final seed size and the size of the endosperm. In common with most dicots, endosperm is not present in the mature Arabidopsis seed but is required to nourish the developing embryo. Therefore, increased endosperm size translates into increased seed size by increasing embryo size, presumably by accumulating and then supplying increased nutrition, or by some other less direct means enabling the embryo to accumulate more resources from the mother.
In wild type 2x-2x crosses the endosperm is triploid and is formed by the fertilisation of a pair of fused haploid polar nuclei of maternal origin with a haploid sperm of paternal origin. Consequently, there is a 2:1 ratio of maternal to paternal genomes in the normal endosperms. An excess of paternal genomes in the endosperm, e.g. as a result of a 2x4x cross, causes increased endosperm proliferation (hyperplasia). An excess of maternal genomes in the endosperm (4x-2x crosses) has the opposite effect: decreased endosperm proliferation (hypoplasia).
Scott et al (1998) explain these observations in terms of the genomic imprinting (inactivation) of genes that contribute to endosperm vigour, either positively or negatively. Accordingly, paternal gametes have an overall positive effect on endosperm growth because genes that inhibit endosperm growth or functionality are imprinted, whilst genes that have a positive effect escape imprinting and are active in the endosperm. Adding more paternal genomes into the endosperm via a tetraploid pollen parent therefore increases the number of stimulatory genes resulting in a larger endosperm. Maternal genomes have the opposite effect. Importantly, imprinting effects have been recorded in a wide range of plant species including maize and brassicas. In marnmals, a number of genes that influence foetal growth (typically expressed in the placenta) also exhibit uniparental expression due to imprinting during gametogenesis. Extra doses of these genes also have dramatic effects on embryo size.
Hybridisation is recognised as an important process for producing offspring having a combination of desirable traits from both parents. Hybridisation may be interspecific or intraspecific. Interspecific hybridisation is used for introducing desirable traits such as disease resistance into crop species. However, the ability to make successful sexual crosses is frequently restricted to closely related species because of the existence of a variety of pre-fertilisation and post-fertilisation reproductive barriers (see Stoskopf, Tomes and Christie, 1993). One type of post-fertilisation barrier is associated with poor or disrupted endosperm development (post-fertilisation endosperm development barrier), which results in non-viable seed (see Ehlenfeldt and Ortiz, 1995). Endosperm failure in unsuccessful crosses is due to the operation of a genetically determined system known as endosperm dosage (Haig and Westoby, 1991). Endosperm dosage is a form of genomic imprinting. The removal of the endosperm dosage barrier to sexual interspecific hybridisation would have economic benefits, since non-sexual techniques for hybridisation e.g. somatic hybridisation are costly and difficult.
The endosperm dosage system may also prevent intraspecific hybridisation where the parents are of different genomic constitutions (ploidies) (Haig and Westoby, 1991).
The occurrence of successful intra- and interspecific hybridisation can also be problematic. In particular, hybridisation between genetically modified crop plants and non modified cultivated or wild plants thereby creating hybrids carrying transgenes with the potential for environmental and other damage inherent in this form of “transgene escape”, has caused alarm within both the public and the regulatory authorities.
There are various strategies that might be used to prevent transgene escape from crops into the wider environment. Critically, a range or spectrum of methods should be available to meet practical constraints imposed by the requirements of plant breeders and seed producers and the life histories of specific crop species when in the hands of farmers. For example, the complete elimination of flowering is acceptable in vegetable crops and forage grasses during the ‘cropping stage’, but unless this trait is conditional in some way, the production of seed by the seed producer, or the breeding of new varieties by the plant breeder, is rendered difficult or impossible.
In crops where the harvest is a fruit or a seed, given that most crop species are self-pollinating, the production of pollen, by at least the majority of flowers, is essential. Most of the major crops fall into this category.
Cleistogamous plants produce flowers that develop normally but which fail to open. Consequently, self pollination occurs, but no pollen escapes from the flower. Whilst this the implementation of this solution would ‘only’ require modifications to flower design, such an approach might be criticised on the grounds that pollen could escape from damage flowers.
The production of viable sexual hybrids occurs within species (intra-specific hybridisation) or between species (inter-specific hybridisation). However, in the case of inter-specific hybridisation, a successful outcome—viable hybrid seed—is usually only possible between closely related species. Two main barriers prevent hybridisation between more widely diverged species—inter-specific incompatibility at the stigma surface or within the style, which prevent fertilisation, and post-fertilisation barriers which cause seed abortion, usually through failures in endosperm development (Brink and Cooper, 1947; Ehlenfeldt and Ortiz, 1995).
Brink and Cooper (1947) working in Lycopersicum were the first to propose that the primary reason for the failure of inter-specific crosses was the same as for intraspecific crosses, namely failure of the endosperm itself. The operation of this type of barrier has been reported in numerous species including the Brassicas (see Haig and Westoby, 1991). These authors and others (see Ehlenfeldt and Ortiz, 1995) also proposed that endosperm failure in inter-specific crosses is due to an effective, rather than actual, imbalance in the normal ratio of maternal to paternal genomes in the endosperm. Different species are proposed to have different genomic strengths. Hence a cross between plants of the same ploidy may fail because the relative genomic strengths of their respective genomes result in a lethal effective genomic imbalance within the hybrid endosperm. Likewise a cross between two plants of different ploidies may succeed provided their relative genomic strengths result in a hybrid endosperm with a balanced genomic constitution. The setting of genomic strength is proposed to involve genomic imprinting, although the exact nature of the relationship is not understood.
In summary, the failure of intraspecific (interploidy) crosses and crosses between species may have a common cause—a genomic imbalance within the endosperm mediated by genomic imprinting. Modifying the genomic strength of one or both of a pair of species that normally hybridise may have application in generating a lethal relative endosperm imbalance, thereby creating a post fertilisation barrier between the two species. The same approach may have application in providing a post-fertilisation barrier within a species, for example between genetically-engineered crop varieties and non-engineered varieties. Practically, for transgene containment the genomic strength of the crop could be modified to prevent cross hybridisation with any problematic close relatives. Such a technology would facilitate the exploitation of genetically modified plants, with considerable economic and environmental benefits.
There is currently considerable research effort to develop transgenic technologies (see Koltunow et al., 1995) to introduce apomixis into crop species. In natural apomictic plant species 2n seed is produced without fertilisation of the egg. The genetic constitution of the embryo is therefore identical to that of the seed parent. The economic benefits of introducing an apomixis system into crop species include true breeding F1 hybrids. Currently, F1 hybrid seed is produced annually by hybridising two genetically distinct parents in a labour intensive and costly process. True breeding (apomictic) F1 hybrids could be propagated for sale without the hybridisation step. The removal of this step would potentially therefore reduce production costs.
An essential aspect of apomixis is that the embryo is derived from a cell with an unreduced (2n) number of chromosomes. In natural apomicts this is achieved by modifying meiosis (meiotic reconstitution) such that 2n gametes are produced, or deriving the embryo from a somatic cell with the 2n number of chromosomes. Irrespective of the origin of the embryo the endosperm is invariably derived via meiosis which is either restitutional or reductional. In the former case the two polar nuclei, which upon fertilisation produce the endosperm, are 2n and in the later case n. Given that natural apomicts utilise endosperms generated in this way it is likely to be the case for genetically engineered apomictic crop plants.
A potential problem in the development of apomictic crop species, given this likely dependency on ‘sexual endosperms’ (formed by fertilisation), is ensuring the successful development of the endosperm, since the endosperm is required to nourish the embryo or itself represents the principal economic harvest. One barrier to endosperm development is the endosperm dosage system. In species with an endosperm dosage system the ratio of maternal to paternal genomes in the endosperm is 2.1. Deviation from this ratio results in endosperm abortion and seed lethality (Haig and Westoby, 1991). Natural apomicts have adopted a number of strategies to ensure endosperm development. A few species (autonomous apomicts) develop a gynogenetic endosperm (maternal) in the absence of fertilisation of the polar nuclei. The majority however, retain fertilisation of the polar nuclei and maintain a 2:1 genomic ratio by modification of either male meiosis (to produce unreduced gametes) or the fertilisation process e.g. fertilisation involves only 1 polar nucleus. Still other species successfully deviate from the genomic 2:1 ratio.
For engineered apomixis the most attractive solution for ensuring endosperm development is the provision of autonomous endosperm development. Solutions involving fertilisation of the polar nuclei are likely to complicate the delivery of apomixis, for example by necessitating the introduction of a mechanism to prevent fertilisation of the “egg” or the need to devise ways to produce 2n male gametes, or by some other means ensure a 2:1 genomic ratio.
One approach to developing an autonomous apomict involves the induction and isolation of mutant genes that condition endosperm development in sexual species without fertilisation. Extensive screening efforts in Arabidopsis met with limited success having identified several mutant genes that condition only limited endosperm development in the absence of fertilisation (Ohad et al., 1996; Chaudhury et al., 1997; Ohad et al., 1999; Kiyosue et al., 1999; Luo et al., 1999). One potential explanation is that these mutations trigger endosperm development but do not overcome the effects of the endosperm dosage system. Endosperms in the mutants would have a genetic constitution of 2 maternal:0 paternal genomes, which deviates from the normal 2:1 genomic ratio. Significantly, Scott et al, 1998, recently showed that Arabidopsis possesses a dosage system capable of causing seed abortion where the ratio of parental genomes in the endosperm deviates significantly from 2:1.
Autonomous apomixis would enable the crop to produce seed without any requirement for pollen. Hence transgene escape through pollen could be prevented by arranging for the crop plant to carry any form of male sterility that stops the production or release of functional pollen.
The interploidy cross effect on seed size, the post-fertilisation endosperm development barrier to interspecific hybridisation and the barrier to autonomous endosperm development are all explicable in terms of genomic imprinting.
In mammals, a number of genes that influence foetal growth (typically expressed in the placenta) exhibit uniparental expression due to genomic imprinting during gametogenesis. Extra doses of these genes can have dramatic effects on embryo size (Solter, 1998). Genomic imprinting also prevents the development of gynogenetic or androgenetic (two paternal genomes, no maternal genome) embryos (Solter, 1998).
In mammals, genes selected for imprinting are maintained in an inactive state by DNA methylation. The enzyme responsible is DNA methyltransferase (MET) which is encoded by a single gene. Mice embryos containing an inactive DNA methyltransferase gene die at an early developmental stage and express both parental copies of genes that are normally imprinted (i.e. uniparentally expressed) (Li et al, 1993). This demonstrates the involvement of DNA methyltransferase in genomic imprinting and a requirement for imprinting in normal development.
In plants the imprinting mechanism is unknown. However, plant genomes contain relatively large amounts of the modified nucleotide 5-methylcytosine (Gruenbaum et al, 1981). Despite evidence implicating cytosine methylation in plant epigenetic phenomena, such as cosupression and inactivation of transposable elements (Napoli et al, 1990; Bender et al, 1995; Brutnell and Dellaporta, 1994, Martienssen et al ., 1995; Matzke and Matzke, 1995 ) the role of cytosine methylation in plant developmental processes and genomic imprinting remains unclear.
To date three different genes have been found that may be imprinted in the maize endosperm: tubulin (Lund et al 1995 ), a storage protein regulator gene dzr (Chaudhuri, and Messing, 1994) and the r gene transcription factor that regulates anthocyanin biosynthesis (Kermicle and, Alleman, 1990). In each case, the maternally inherited allele is undermethylated, over-expressed or both, whereas the paternally inherited allele is more methylated or has a reduced level of expression.
In Arabidopsis, ddm mutants (decrease in DNA methylation) have been isolated with reduced levels of cytosine methylation in repetitive sequences, although the mutations do not result in any detectable change in DNA methyltransferase activity (Vongs et al., 1993; Kakutani, 1995). After several generations of self pollination, ddm mutants exhibit a slight delay (1.7 days) in flowering, altered leaf shape, and an increase in cauline leaf number (Kakutani, et al, 1995). Repeated self pollination of ddm mutant plants does however result in the appearance of severe developmental abnormalities (Kakutani et al, 1996).
Arabidopsis plants expressing DNA methyltransferase 1(Met1) antisense (Met 1as) gene contain reduced levels of DNA methyltransferase activity and a correspondingly reduced level of general DNA methylation (Finnegan et al., 1996; Ronemus et al., 1996). In contrast to ddm mutants, Arabidopsis plants expressing a Met1 as gene develop various developmental abnormalities at high frequency and without repeated self-fertilization, including floral abnormalities (Finnegan et al., 1996). PCT/US971/13358 also reports that Arabidopsis plants expressing a Met1as gene alter the rate of development of the plant. The development of the endosperm in ddm mutants and plants expressing Met1as has not been reported.
The present invention is based on the unexpected observation that a decrease of about 90% in the amount of methylated DNA present in a plant genome results in the production of gametes, both male and female, that behave in a manner that is consistent with the removal or attenuation of genomic imprinting. This is exemplified by the following experiments:    1. Endosperm development in seeds derived from a cross between a wild type 2x plant, as seed parent, and a 2x Met1as plant as pollen parent (2x-2xMet1as), resembles endosperm development in seeds derived from a 4x-2x interploidy cross (FIGS. 1 and 3).—the endosperm is small/underdeveloped. The resulting seed is smaller in weight terms than seed from control 2x-2x crosses (Table 1). Hence the male gametes from a Met1as plant behave like a female gamete from a wild type plant. This can be explained by proposing the removal or attenuation of imprinting in the male gamete.    2. Endosperm development in seeds derived from a cross between a 2xMet1as plant, as seed parent, and a wild type 2x plant as pollen parent, strongly resembles endosperm development in seeds derived from a 2x-4x interploidy cross between wild type plants (FIGS. 1 and 3).—that is, the endosperm is large/overdeveloped. The resulting seed is larger in weight terms than seed from control 2x-2x crosses (Table 1). Hence the female gametes from a 2xMet1as plant behave as a male genome of a normally methylated diploid plant. This can be explained by proposing the removal or attenuation of imprinting in the female gamete.    3. Reciprocal crosses between 2xMet1as and 4x wild type plants result in seed abortion (FIGS. 1 and 3); consequently seeds derived from these crosses are shrivelled and do not germinate (Table 1). The behaviour of the endosperm in seed generated in these crosses depends on the direction of the cross. Where the 4x plant is the seed parent the endosperm is extremely under-developed and contains very few endosperm nuclei and a very small chalazal endosperm (FIG. 1, Table 1). In contrast, where the 4x plant is the pollen parent the endosperm of the resulting seeds is over-developed, and contains many endosperm nuclei and a very well developed chalazal endosperm with many associated chalazal nodules (FIGS. 1 and 3, Table 1). This outcome resembles those obtained in crosses between 2x and 6x wild type plants which routinely fail to produce viable seed (FIG. 3) and display very under—(6x-2x) or over-developed (2x-6x) endosperm depending on the direction of the cross. These crosses represent examples of lethal parental genomic excesses within the endosperm that result from the large disparity between the ploidy level of the respective parents. The similarity between the outcomes and the behaviour of the endosperm in 2xMet1as—4x and 2x-6x reciprocal crosses can be explained by proposing that male and female gametes derived from 2xMet1as plants behave, in part, like gametes of the opposite sex with respect to genomic imprinting. This again strongly suggests that DNA hypomethylation caused by the Met1as gene removes or strongly attenuates genomic imprinting.    4. The behaviour of plants homozygous for the ddm mutation in reciprocal crosses with 2x and 4x wild type plants is very similar to that of plants homozygous for the Met1as gene (see FIG. 2 and Table 1). This strongly suggests that the basis of the interploidy cross effect is associated with general DNA hypomethylation.